The present invention relates to protecting groups, more particularly to compounds useful for protecting amino, amidino, guanidino and hydroxyl groups, as well as methods of use thereof during oligonucleotide synthesis.
During oligonucleotide synthesis, convenient amino group protection methodology is important not only for exocyclic amines but also for side chain amino groups (xe2x80x9caminolinkersxe2x80x9d or xe2x80x9caminotethersxe2x80x9d). These amino group-containing tethers can be conveniently deprotected and used to attach various functionalities to modify the biological or chemical properties of oligonucleotides (e.g., to conjugate groups which can improve uptake of antisense oligonucleotides by living cells); to attach chemical nucleases targeting the pathogenic genes; and to attach reporter groups (such as fluorescein or biotin) which are extensively used in DNA based diagnostics in following cellular trafficking of antisense oligonucleotides (Manoharan, M., in Antisense Research and Applications, S. T. Crooke and B. Lebleu (eds.), CRC Press, Boca Raton, Fla., 1993, 303-349). In spite of their widespread use, the conventional protecting groups used in oligonucleotide chemistry for aminotethers are either too labile during the monomer synthesis (e.g., CF3COxe2x80x94, Fmoc) or somewhat inert, thereby requiring harsh conditions during, for example, oligonucleotide deprotection. The phthalimido group, for example, requires CH3NH2 in addition to the standard ammonium hydroxide conditions. The acid labile MMT (monomethoxytrityl) group is sometimes used, but generally to protect an aminolinker only at the 5xe2x80x2-end of the oligonucleotide.
To overcome these problems the alloc (allyloxycarbonyl) group (Kunz. H. Angew. Chem. 96, 426, 1984; Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J. Am. Chem. Soc. 112, 1691, 1990) has been adopted as a protecting group for aminolinkers, as it can be removed using zerovalent palladium (Pd (0)) either in solution phase or in solid phase (Barber-Peoch, I; Manoharan, M.; Cook, P. D. Nucleosides and Nucleotides 16, 1407-1410, 1997; Nelson, P. S.; Muthini, S.; Kent, M. A; Smith T. H.; Nucleosides and Nucleotides 16, 1951-1959, 1997). The chloroformate Clxe2x80x94(Cxe2x95x90O)xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94CN also has been used to protect nucleobase amines. Chloroformates, however, are unstable and difficult to use. It would therefore be desirable to provide alternative reagents for protecting amine and other groups during synthesis.
In one aspect, the present invention provides compounds of the formula (I) 
wherein
X is aryl or a covalent bond;
Y is aryl or a covalent bond;
R1 is selected from succinimid-N-yl, phthalimid-N-yl, pyridin-N-yl, 4-nitophenyl, N-imidazol-1-yl, benzotriazol-2-yl, pyridin-2-yl, pentafluorophenyl, tetrafluorophenyl, triazol-N-yl, tetrazol-N-yl and norbornan-N-yl;
R2 is cyano, nitro or CF3;
R3 and R4 are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl;
W is C(R5) (R6) or C(R7)xe2x95x90C(R7) where each R5, R6, and R7 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl or both R7 substituents together form an unsaturated aromatic ring; and
n is an integer from 0 to 7.
In another aspect, the invention provide methods for protecting amine, guanidine, amidine or hydroxyl groups comprising reacting a free amine, guanidine, amidine or phosphate with a compound according to the general formula (I).
R1 can be selected from succinimid-N-yl, phthalimid-N-yl, pyridin-N-yl, 4-nitophenyl, -imidazol-1-yl, benzotriazol-2-yl, pyridin-2-yl, pentafluorophenyl, tetrafluorophenyl, triazol-N-yl, tetrazol-N-yl, pyrazol-N-yl and norbornan-N-yl. More preferably, R1 is succinimid-N-yl, phthalimid-N-yl or pyridin-N-yl, and even more preferably succinimid-N-yl or phthalimid-N-yl. Most preferably, R1 is succinimid-N-yl.
R2 can be cyano, nitro or CF3. In a preferred embodiment, R2 is cyano while X and Y are both a covalent bond. In another preferred embodiment R2 is nitro while one of X and Y is aryl while the other is a covalent bond. In another embodiment R2 is cyano while Y is aryl (e.g., 1,4-phenylene) and X is a covalent bond.
R3, R4, R5, R6, and R7 each be independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl and cycloalkyl-alkyl.
As used herein, the term xe2x80x9calkylxe2x80x9d includes but is not limited to straight chain, branch chain, and cyclic unsaturated hydrocarbon groups having 1 to about 10 (preferably 1 to about 4) carbon atoms. xe2x80x9cAlkenylxe2x80x9d includes but is not limited to straight chain, branch chain, and cyclic saturated hydrocarbon groups having 2 to about 10 carbon atoms. xe2x80x9cAlkynylxe2x80x9d includes but is not limited to hydrocarbon groups having 2 to about 10 carbon atoms and a carbonxe2x80x94carbon triple bond. By xe2x80x9ccycloalkylxe2x80x9d is meant mono- or bicyclic rings of 3 to 10 members optionally unsaturated and optionaly attached to the carbon atom from which R3 to R7 depend by an alkyl chain thereby forming a xe2x80x9ccycloalkylxe2x80x9d group. When any of R3 through R7 is alkenyl or alkynyl, the unsaturated bond or bonds preferably are spatially removed from the carbon atom from they depend. In other words, the unsaturated bond is preferably not adjacent to said carbon atom.
xe2x80x9cArylxe2x80x9d is used herein interchangeably with xe2x80x9caromaticxe2x80x9d, and includes optionally substituted mono-, bi- and tricyclic, 5 to 14 membered rings incorporating carbon atoms exclusively or incorporating one or more heteroatoms such as N, O and S (as well as SO and SO2), thereby forming a heteroaryl group. Prefered aryl groups include phenyl, naphthyl, pyridyl, quinolinyl, isoquinolinyl and naphthyridyl.
In preferred embodiments, R3 through R7 are independently H, alkyl or aryl. In a particularly prefered embodiment R3, R4 and R5 are H and R6 is phenyl (preferably while n is 1). In another particularly prefered embodiment, both R3 and R4 are H and both R5 and R6 are methyl (preferably while n is 1). In another preferred embodiment, each of R3 through R6 are H (preferably while n is 1).
In other embodiments in which W includes olefinic moieties, R2 preferably is cyano or nitro. When W is olefinic, adjacent R7 substituents together may form an aromatic ring. Prefered aromatic (i.e., aryl) rings formed in this respect include benzene, naphthalene, pyridine and more preferably benzene.
X and Y preferably are both covalent bonds. In an alternate embodiment, one of X and Y is aryl (e.g., 1,4-phenylene) wherein X and CR5R6 are para to one another.
Compounds of the invention can be prepared according established organic synthetic techniques. In a particular general method, compounds are prepared by reacting an alcohol of formula (II) under suitable conditions (e.g. in acetonitrile/dichloromethane in the presence of pyridine) with a dicarbonate of formula (III), wherein X, Y, n and R1 through R7 are as previously defined. See Scheme 1 below. The alcohol (II) and dicarbonate (III) are either commercially available or themselves are prepared from commercially available reagents according to established synthetic techniques. 
Alternatively, compounds of formula (I) can be prepared by reacting a chloroformate of formula (IV) under suitable conditions with an alcohol of formula (V). See Scheme 2 below. 
Again, chloroformate (IV) and alcohol (V) are either commercially available or themselves may be prepared from commercially available reagents using established organic synthetic techniques.
The invention also provides methods for protecting amine, guanidine, amidine or hydroxyl groups comprising reacting a free reactive amine, guanidine, amidine, or hydroxyl with a compound according to the general formula (I). Said amine, guanidine, amidine or hydroxyl group may be incorporated on or within various chemical molecular entities requiring protection, including but not limited to amino acids, peptides, proteins, nucleosides (RNA or DNA), nucleotides and oligonucleotides. In particular, amino acids incorporating amines and guanidines in their side chains (e.g., lysine and arginine, respectively) are suitable for methods of the invention. Other compounds particularly amenable to the present invention are nucleosides, nucleotides and oligonucleotides. For instance, exocyclic amine groups on nucleobases (e.g., cytosine, adenine and guanine) can be protected according to methods of the invention. A class of nucleosides known as xe2x80x9cpeptide nucleic acidsxe2x80x9d (PNAs) incorporate a peptidic backbone in place of a natural sugar-phosphate backbone, the amine group of which may also be protected according the method of the invention. Further, hydroxyl groups on the sugar of a nucleoside (e.g., at the 2xe2x80x2-, 3xe2x80x2- and 5xe2x80x2-positions) also can be protected.
In a particular embodiment, the amine, amidine, guanidine and hydroxyl groups that are protected according to the present invention are located on tethering or linker groups. The amine, amidine, guanidine or hydroxyl groups on these tethers can, upon deprotection, be used to attach functional groups to nucleosides, nucleotides, and, in particular, oligonucleotides to modify the biological or chemical properties of such moieties. Functional groups include conjugate groups (such as cholesterol) for enhancing cellular uptake, intercolators for enhancing hybridization, chemical nucleases for targeting pathogenic genes, and reporter groups (such as fluorescein and biotin) for diagnostic purposes or monitoring cellular trafficking. Tethers can be attached to the nucleobase, backbone or sugar moieties. In a particular embodiment, tethers are attached at the 2xe2x80x2-O position of a nucleoside sugar. Alternatively, tethers may be attached at the 3xe2x80x2-O position, for example when oligonucleotides have one or more 2xe2x80x2-5xe2x80x2 linkage or at the 5xe2x80x2-O position when the tether is at the 5xe2x80x2 terminus of an oligonucleotide.
Protecting groups may be subsequently removed to give the free amine, amidine, quanidine or hydroxy by reacting with a suitable reagent. For amines, amidines and guanidines, suitable removing agents include ammonium hydroxide (NH4OH), triethylamine (NEt3), DBU, Hunig""s base, (iPr)2NEt, Et2N4, piperidine, morpholine, piperazine and pyrrolidine. For hydroxyl groups, suitable reagents for removing protecting groups include DBU. Advantageously, one can achieve deprotection of all amine groups (nucleobase and tethered amines) and all phosphates of an oligonucleotide synthesis in a single step. This is accomplished when a suitable phosphate protecting group is employed such as xcex2-cyanoethyl and a suitable deprotection reagent is used such as ammonium hydroxide. Alternatively, the protecting group may be employed at specified groups and not at others, thereby affording selective protection/deprotection ability.
In one aspect of the invention, compounds of formula (I) are used in a guanylating process. A thiopseudourea of formula (VI) (wherein Rxe2x80x2 is a suitable protecting group such as alkyl, e.g. methyl) is reacted with a dicarbonate of formula (III) to give a mixture of mono and bis protected thiopseudourea (VII) and (VIII) (wherein Q is xe2x80x94Xxe2x80x94(CR5R6)nxe2x80x94CR3R4xe2x80x94Yxe2x80x94R2). The bis compound (VIII) is then reacted with a primary amine RNH2 wherein R is H or a guanidino protecting group such as alkyl (e.g., methyl) or BOC to give a bis protected guanidino group (IX). Guanidino (IX) may then be used to add a guanidino functionality to other compounds, in particular those with a reactive hydroxyl group to give a protected functional group xe2x80x94NHxe2x80x94C(NH)xe2x80x94NHxe2x80x94COxe2x80x94Oxe2x80x94(CR5R6)Nxe2x80x94CR3R4xe2x80x94Yxe2x80x94R2. Deprotection of the guanidinyl group may achieved using a suitable reagent including base. 
Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting.